drought tolerance in plants

ABSTRACT

The invention provides methods for enhancing drought tolerance in a plant, by infecting the plant with a plant virus, or infectious material derived from a plant virus. The plant thereby displays fewer, less severe, and/or delayed symptoms of dehydration. The reduction in symptoms allows for improved plant growth.

This application claims the priority of U.S. Provisional PatentApplication 60/763,668, filed Jan. 30, 2006, which is hereinincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of agriculture.More particularly, the invention relates to the use of plant viruses forimproving drought tolerance in plants.

2. Description of Related Art

Plant viruses are often discovered and studied as pathogenic parasitesthat cause disease in plants, including agricultural crops. Numerousplant viruses and virus strains are known, and may be classified basedon the structure of their encoding nucleic acids (e.g. DNA or RNA), itspolarity (positive or negative sense), genome strandedness, genomestructure (e.g. monopartite, bipartite), and on capsid structure (e.g.icosahedral, rigid rod, flexuous rod, etc.). For a positive-sense virus,the genome and the mRNA correspond to the same nucleic acid strand,while for a negative-stranded virus, the genome corresponds to thecomplement of the viral mRNA. The majority of plant viruses arepositive-sense single-stranded RNA viruses. Important groups of plantviruses include the Bromoviruses, Potyviruses, Tobamoviruses, andCaulimoviruses, among others. Virus satellites and viroids are alsoknown, which are infectious nucleic acids that depend on the function ofa structurally unrelated helper virus for their replication.

Infection of a plant host by a compatible virus, and subsequentreplication and spread in the plant often results in symptoms includingaltered leaf color (e.g. chlorosis, mosaic, or vein clearing), stunting,reduced yield, and altered morphology of plant structures, among others.Such viruses may spread systemically throughout a plant from the initialinfection site. Incompatible viruses, in contrast, replicate and/orspread poorly, if at all, in a plant. Some plant hosts are asymptomatic,and may serve as reservoirs leading to infections that are onlyrecognized on symptomatic hosts. It is widely believed that viruses areharmful to their hosts because they use host resources to support theirown reproduction, and may interfere with plant metabolism such aschloroplast development or function.

Plant responses to biotic and abiotic stress, including abiotic stresssuch as drought (e.g. Bohnert et al., 1995; Bray, 1997), are complex,and are thought to include multiple signaling pathways including changesin levels of protective osmolytes and antioxidants, stress proteins, andchanges in levels of plant hormones, among other responses. TMVinfection of host plants has been reported to result in increasedabscisic acid (ABA) levels in infected tobacco plants (Whenham et al.,1986).

For some bacterial, fungal, and animal viruses mutualistic relationshipshave been described. For instance, some ascoviruses of wasps can bemutualistic depending on the specific virus and wasp strains (Stasiak etal., 2005). In addition, the polydnaviruses of braconid wasps arerequired for survival of the parasitoid wasps in their caterpillar hosts(Webb, 1998). Several dsRNA mycoviruses are known in the fungus Ustilagomaydis. For instance, the virus UMV4 encodes KP4 killer toxin, andenables its host, U. maydis strain P4, to outcompete other uninfected U.maydis strains and related fungi (Gage et al., 2001). Human endogenousretroviruses may protect human tissue from infection with the exogenousretrovirus Spleen necrosis virus and may protect a developing fetus(Ryan, 2004). Certain mutualistic symbioses have also been reported inplants (e.g. Schardl et al., 2004). However, mutualistic interactionsbetween plant viruses and their hosts have not been previouslydescribed, nor has the use of virus infection for improving droughttolerance in plants. Such interactions may have important agriculturalapplications, especially as drought is one of the most limiting factorsin crop production worldwide (Wollenweber et al., 2005).

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for enhancing droughttolerance in a plant comprising: (a) identifying a plant in need ofincreased drought tolerance; and (b) infecting the plant with a plantvirus or infectious plant viral material that increases the droughttolerance in the plant as compared to an uninfected plant of the samegenotype when grown under the same conditions. In the method the virusmay or may not produce a disease symptom in the plant. In oneembodiment, step (a) comprises identifying a plant exhibiting droughtstress. In another embodiment, step (a) comprises identifying a plant ina growing environment under drought conditions.

A plant used with the invention may be any plant and in certainembodiments may be defined as a rice, beet, cucumber, zucchini,watermelon, tomato or Nicotiana sp. In particular embodiments, a virusused with the invention may be Cucumber Mosaic Virus (CMV), and may beselected from the group consisting of: Cucumoviruses, Tobamoviruses,Tobraviruses, and Bromoviruses, including a virus is selected from thegroup consisting of Cucumber Mosaic Virus, Tobacco Mosaic Virus, TobaccoRattle Virus, and Brome Mosaic Virus. An infectious material may be usedin accordance with the invention, such as a plant virus virion, an RNAtranscript, or a plant virus cDNA clone. In certain embodiments, a plantmay be infected with a virus by a method selected from the groupconsisting of: mechanical inoculation, spraying, injection,infiltration, grafting, seed or pollen transmission, and vectortransmission. Virus infection may comprise mechanical inoculation.

In another aspect, the invention provides a method of reducing osmoticstress damage in a plant, comprising the steps of: (a) identifying aplant under osmotic stress; and (b) infecting the plant with a plantvirus.

The foregoing has outlined certain features and technical aspects of thepresent invention in order that the detailed description of theinvention that follows may be better understood. Additional features andadvantages of the invention will be described hereinafter which form thesubject of the claims of the invention. It should be appreciated bythose skilled in the art that the conception and specific embodimentdisclosed may be readily utilized as a basis for modifying or designingother structures for carrying out the same purposes of the presentinvention. It should also be realized by those skilled in the art thatsuch equivalent constructions do not depart from the spirit and scope ofthe invention as set forth in the appended claims. The novel featureswhich are believed to be characteristic of the invention, both as to itsorganization and method of operation, together with further objects andadvantages will be better understood from the following description whenconsidered in connection with the accompanying figures. It is to beexpressly understood, however, that each of the figures is provided forthe purpose of illustration and description only and is not intended asa definition of the limits of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: Comparison of drought symptoms in mock- and CMV-infected riceplants at 7 days after watering.

FIG. 2: Comparison of drought symptoms in mock- and BMV-inoculated riceplants at 6 days after watering.

FIG. 3: Comparison of drought symptoms in mock- and TMV-inoculated N.benthamiana plants at 8 days after watering.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are methods and compositions for improving stresstolerance in plants. The methods enhance the ability of plants towithstand stress such as drought. Surprisingly, it was found thatsystemic infection of a host plant by a symptomatic or asymptomaticplant virus improves the growth and/or survival of the host underdrought conditions. As used herein, “drought” refers to a condition inwhich a plant is subjected to osmotic stress or reduced water potential.

Although plant viruses were initially identified and studied based ontheir ability to cause disease, as disclosed herein this previousparadigm represents an incomplete picture of the virus-hostrelationship. Rather, it is demonstrated that mutualistic relationshipsmay be established between a plant pathogenic virus and its symptomaticor asymptomatic host plant under drought stress. In this instance, theappearance of drought symptoms may be delayed and/or their severity maybe reduced in a virus-infected plant as compared to uninfected plants ofthe same species, line, or cultivar, under the same environmentalconditions.

The present invention provides methods for improving drought(dehydration) tolerance in plants subject to water limiting conditionscomprising introducing a plant virus into a plant. The plant virus mayinduce a viral disease symptom upon its introduction, yet infectionresults in enhanced drought tolerance. Alternatively, no symptoms ofviral disease may be induced, however drought tolerance is neverthelessenhanced. Drought tolerance may be assessed by comparing a virusinfected plant with a non-infected plant of the same genotype, variety,or cultivar grown under the same conditions of drought or limited wateravailability. One or more symptoms may be compared between infected anduninfected plants, including, for instance, wilting, growth arrest, leafrolling, leaf distortion, leaf drop, leaf scorch, stem or twig dieback,photosynthetic efficiency, flowering, and yield level.

Drought tolerant plants demonstrate fewer or less severe symptoms ofstress caused by lack of available water. Drought conditions, or lack ofavailable water, may be assessed by comparing the amount of waterrequired for plant growth or maturation with the amount of wateravailable to a plant. Drought conditions may be caused by lack ofrainfall or irrigation, relative to the amount of water used internallyor transpired by a plant. Physiological parameters such as waterpotential may be measured to quantify a plant's level of water stress.Phenotypic assessment of symptoms described above may be utilized todetermine whether, and to what extent, a plant is suffering fromdrought. Alternatively, biochemical or nucleic acid based assays may beperformed to assess a plant's response to water limitation.

A plant or plant tissue may be inoculated or infected with a virus orinfectious viral material by any suitable method for delivering thematerial to the plant, such as by mechanical inoculation or by spraying,by injection or infiltration, by grafting, by seed or pollentransmission, or by vector transmission (e.g. by an insect or nematodevector), among others. Inoculation may be performed, for example, into awound or in the presence of an abrading agent. Spray-inoculation mayalso be employed, optionally in conjunction with abrasion of planttissues.

The infectious material may comprise purified, partially purified, orunpurified nucleic acids or viral particles, including an infectiousviral DNA or cDNA clone, infectious RNA transcript(s), virions, sap frominfected plants, ground leaves, or other tissue or tissue extract froman infected plant. By “infectious”, it is meant that the material allowsfor viral replication within a plant cell.

The infectious material may be suspended or diluted in a physiologicallyacceptable carrier, solvent, or diluent, such as a phosphate buffersolution (e.g. at a pH of 7.0-7.4), or other dispersion media, and thelike. An inoculum-containing solution may also be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose, and/oran abradant such as Carborundum™ or silicon carbide. Dispersions alsocan be prepared in a solution comprising glycerol, or a polyol andmixtures thereof. The infectious material may be in the form of asolution, an aerosol, a dispersion, a powder, or the like. A virusstrain selected for inoculation may be genetically engineered with acoding or non-coding sequence, for instance to allow for specificdetection of the inoculated strain, for instance by nucleic acidamplification or hybridization, by antigen detection, or by activity ofa marker protein such as GFP (Green Fluorescent Protein).

The tested plant virus strains resulted in improved drought toleranceunder the conditions used. Thus, the invention is not limited to anyspecific plant virus strain. In certain embodiments, the virus utilizedfor plant infection may be an RNA virus such as a Cucumovirus, aTobamovirus, a Tobravirus, or a Bromovirus. In particular embodiments,the virus may be a strain of: Cucumber Mosaic Virus (CMV), TobaccoMosaic Virus (TMV), Tobacco Rattle Virus (TRV), or Brome Mosaic Virus(BMV), among others. In other embodiments, other RNA viruses may be usedand in a further embodiment a DNA virus is used according to theinvention.

In one embodiment of the invention, the plant may be a monocot cropplant, such as rice. In another non-limiting embodiment of theinvention, the plant may be, for example, a dicot crop plant such astobacco, cucumber, tomato, pepper, beet, watermelon, zucchini. The plantmay also be an ornamental plant, such as poinsettia.

The invention also provides a method for altering water consumption by aplant, wherein a virus infected plant requires or consumes less waterthan an uninfected plant of the same genotype, variety, or cultivar inorder to achieve comparable survival, growth, flowering, and/orproduction of harvestable material. The invention further provides amethod to reduce osmotic stress damage in a plant, by selecting a plantunder osmotic stress or at risk for osmotic stress, and infecting theplant with a plant virus, such that the plant displays fewer, lesssevere, and/or delayed symptoms of water deficiency relative to anuninfected plant.

Techniques for nucleic acid and protein detection may find use incertain embodiments of the invention. For example, such techniques mayfind use in testing for the presence of a virus in a plant that may beinfected with a virus.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Viruses and Plant Materials

The Russian strain of Brome mosaic virus (BMV) and the U1 and mutantMIC-1,3-strains of Tobacco mosaic virus (TMV) were used (Shintaku etal., 1996) for BMV and TMV inoculations, respectively. For Tobaccorattle virus (TRV) infection, a cDNA clone derived from, and containinga portion of the GFP gene, was used (Ryu et al., 2004). The Fny strainof Cucumber mosaic virus (CMV), previously described (Xu et al., 2004),was used for CMV inoculation. Inoculum consisted of purified virus (CMV)or sap from infected plants (BMV, TMV and TRV). For CMV or TMV inoculumwas prepared with purified TMV particles in pH 7.2 PBS (0.05 mM) at aconcentration of 200 ng/μl and 10 μl of inoculum was placed on the leafsof tested plants. For sap inoculation infected N. benthamania plantswere ground in PBS buffer (pH 7.2, 50 mM) and 40 μl sap was inoculatedonto leafs of tested plants by mechanical grinding.

Seedlings at the 2-3 leaf stage were used for inoculation of beet (Betavulgaris cv. Detroit Dark Red), pepper (Capsicum annuum cv. Marango),watermelon (Cucumis lanatus cv. Crimson Sweet), cucumber (Cucumissativus cv. National Pickling), tomato (Lycopersicon esculentum cv.Rutgers), Lycopersicon hirsutum, rice (Oryza sativa cv. IR-8) andzucchini (Cucurbita pepo, cv. Elite). One month old seedlings were usedfor inoculation of Nicotiana benthamiana and of tobacco (N. tabacum cv.Xanthi nc for CMV inoculation, and cv. Xanthi nn for TMV inoculation).For TMV infections, N. benthamiana was inoculated with theMIC-1-3-mutant of U1-TMV because U1-TMV is lethal, and tobacco wasinoculated with the U1-strain of TMV.

Example 2 Drought Treatment

For each plant species, eight to fifteen individual plants wereinoculated with buffer or viral inoculum. After inoculation, the plantswere grown in four separate growth rooms. The temperature ranges ofthese growth rooms were 20° C. (night) 26° C. (day) for TMV and CMVrooms, 19° C. (night) 24° C. (day) for the BMV room, and 18° C. (night)23° C. (day) for the TRV room, with 16 hour days. Eight days(watermelon, cucumber, tomato, pepper, C. amaranticolor and L. hirsutumplants) or two weeks (N. benthamiana and tobacco) post-inoculationplants were bottom watered for two days to saturate the soil, and thenmoved to dry flats where water was withheld. The drought-treated plantswere photographed with a COOLPIX990 digital camera (Nikon, Melville,N.Y., USA) once every day (days after withholding water; daw) from theonset of drought symptoms until the death of the mock-inoculated plants.

Example 3 Infection of Plants with Cucumber Mosaic Virus (CMV)

Eleven different plant species were infected with the broad host rangevirus Cucumber mosaic virus (CMV), strain Fny (Roossinck, 1998) (Table1). These plant species included rice, Oryza sativa, which has not beenreported previously as a host of CMV. Inoculum consisted of purifiedCMV. Table 1 lists the plant species inoculated with CMV and theirresponse to drought conditions. The appearance of drought symptoms invirus-infected plants was delayed by 2-5 days compared withmock-inoculated plants. TABLE 1 Time, in days, of the onset of droughtsymptoms and plant death after withholding water for mock-inoculated andCMV-infected plants. Appearance of Occurrence of symptoms plant deathMock CMV Mock CMV Plant species Common name (daw) (daw) (daw) (daw) Betavulgaris Beet 5 8 8-9 11-12 Capsicum annum Pepper 2 6-8 5-6 11-13Chenopodium Lambsquarters 3 6 6  8 amaranticolor Cucumis lanatusWatermelon 5  9-10 9 11-12 Cucumis sativus Cucumber 3 6 5  9-10Cucurbita pepo Zucchini 3 6  9-10 12 Lycopersicon Tomato 1 3 2-3 5-7esculentum Lycopersicon — 7 10-11  9-10 12-13 hirsutum Nicotiana — 2 615-16 18-19 benthamiana Nicotiana tabacum Tobacco 2 4-5  7-10  8-11Oryza sativa Rice 6 8-9 9 11-14

Example 4 Identification of Systemic Infection of CMV

To determine if rice supports CMV infection, the systemic (i.e.non-inoculated leaves) leaf tissues of buffer- or CMV-inoculated riceand tomato plants (positive control) were harvested, and total RNA wasextracted as previously described (Xu et al., 2004). Five μg of totalRNA was used for RT-PCR amplification of CMV RNA3 using forward primer5′ GGATGCGCGCTGATAATGCT 3′ (SEQ ID NO:1) and reverse primer 5′CCGAAGGAATTCCGAAGAAACCTAGG 3′ (SEQ ID NO:2) primers. The amplifiedfragment was gel extracted and analyzed by sequence analysis. Followinginoculation, asymptomatic rice plants were found to support CMVreplication.

To determine if CMV moves out of the inoculated leaves of C.amaranticolor, the inoculated leaves (positive control), the stem abovethe CMV-inoculated leaves and the non-inoculated upper leaves of buffer-or CMV-inoculated C. amaranticolor were harvested and total RNA wasextracted and analyzed as described above.

Example 5 N. benthamiana Inoculations

Nicotiana benthamiana is a common host for the four RNA viruses used inthis study. Thus, N. benthamiana plants were inoculated with theseviruses to compare their effects on drought tolerance. The appearance ofdrought symptoms in virus-infected plants was delayed by 2-5 dayscompared with mock-inoculated plants (Table 2). The TMV-infected plantsexhibited slow but perceptible growth while the mock-inoculated plantssuffered growth arrest soon after the withholding of water began (FIG.3). TABLE 2 Time, in days, of the onset of drought symptoms and plantdeath after withholding water between mock- and virus-infected N.benthamiana plants. MIC-1,3- BMV (daw) CMV (daw) TMV (daw) TRV (daw)Mock BMV Mock CMV Mock TMV Mock TRV Appearance 4 6 2 6 4 7 3 7 ofdrought symptoms Occurrence 21-22 22-24 15-16 18-20 18-19 21-22 16-1819-22 of plant death

Example 6 Inoculation of Rice and Tobacco

Rice seedlings were inoculated with BMV as described above. BMV-infectedrice showed the first sign of drought stress (rolled leaves) 9-10 daysafter withholding water (daw), by which time mock-inoculated rice plantswere completely wilted (FIG. 2). Tobacco seedlings (N. tabacum genotypenn) were inoculated with TMV as described above. TMV-infected tobaccoplants survived with turgid stems and green tips for 50-55 daw, whilemock-inoculated plants died more than twenty days earlier.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods in the steps or inthe sequence of steps of the methods described herein without departingfrom the concept, spirit and scope of the invention. More specifically,it will be apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   Bohnert et al., 1995. Plant Cell 7:1099-1111.-   Bray, 1997. Trends Plant Sci. 2:48-54.-   Gage et al., 2001. Mol. Microbiol. 41:775-785.-   Roossinck, M. J. and P. S. White. 1998. in Plant Virology    Protocols G. D. Foster, S. C. Taylor, Eds. (Humana Press, Totowa,    N.J.), vol. 81, pp. 189-196.-   Ryan, F. 2004. J R Soc. Med. 97:560-   Ryu, et al., 2004. Plant J 40:322-331.-   Schardl, et al. 2004. Ann. Rev. Plant Biol. 55:315-340.-   Shintaku et al., 1996. Virology 221:218-225.-   Stasiak, et al., 2005. J Insect Physiol. 51:103-115.-   Webb, B A, in The Insect Viruses L. K. Miller, L. A. Ball, Eds.    (Plenum Publishing Corporation, New York, 1998) pp. 105-139.-   Whenham et al., 1986. Planta 168:592-598.-   Wollenweber, et al., 2005. Cur. Opinion. in Plant Biol. 8:337-341.-   Xu et al, 2004. Proc. Natl. Acad. Sci. USA 101:15805-15810.

1. A method for enhancing drought tolerance in a plant comprising: (a)identifying a plant in need of increased drought tolerance; and (b)infecting the plant with a plant virus or infectious plant viralmaterial that increases the drought tolerance in the plant as comparedto an uninfected plant of the same genotype when grown under the sameconditions.
 2. The method of claim 1, wherein the virus produces adisease symptom in the plant.
 3. The method of claim 1, wherein thevirus does not produce a disease symptom in the plant.
 4. The method ofclaim 1, wherein step (a) comprises identifying a plant exhibitingdrought stress.
 5. The method of claim 1, wherein step (a) comprisesidentifying a plant in a growing environment under drought conditions.6. The method of claim 1, wherein the plant is selected from the groupconsisting of rice, beet, cucumber, zucchini, watermelon, tomato,tobacco, and Nicotiana sp.
 7. The method of claim 6, wherein the plantis rice (Oryza sativa).
 8. The method of claim 7, wherein the virus isCucumber Mosaic Virus (CMV).
 9. The method of claim 1, wherein the virusis selected from the group consisting of: Cucumoviruses, Tobamoviruses,Tobraviruses, and Bromoviruses.
 10. The method of claim 9, wherein thevirus is selected from the group consisting of Cucumber Mosaic Virus,Tobacco Mosaic Virus, Tobacco Rattle Virus, and Brome Mosaic Virus. 11.The method of claim 1, wherein the infectious material comprises a plantvirus virion, an RNA transcript, or a plant virus cDNA clone.
 12. Themethod of claim 1, wherein the plant is infected with a virus by amethod selected from the group consisting of: mechanical inoculation,spraying, injection, infiltration, grafting, seed or pollentransmission, and vector transmission.
 13. The method of claim 12,wherein the method for virus infection is mechanical inoculation. 14.The method of claim 12, wherein the virus is selected from the groupconsisting of Cucumber Mosaic Virus, Tobacco Mosaic Virus, TobaccoRattle Virus, and Brome Mosaic Virus.
 15. A method of reducing osmoticstress damage in a plant, comprising the steps of: (a) identifying aplant under osmotic stress; and (b) infecting the plant with a plantvirus.